Dynamic Loading and Tendon Healing Affect Multiscale Tendon Properties and ECM Stress Transmission.

The extracellular matrix (ECM) is the primary biomechanical environment that interacts with tendon cells (tenocytes). Stresses applied via muscle contraction during skeletal movement transfer across structural hierarchies to the tenocyte nucleus in native uninjured tendons. Alterations to ECM structural and mechanical properties due to mechanical loading and tissue healing may affect this multiscale strain transfer and stress transmission through the ECM. This study explores the interface between dynamic loading and tendon healing across multiple length scales using living tendon explants. Results show that macroscale mechanical and structural properties are inferior following high magnitude dynamic loading (fatigue) in uninjured living tendon and that these effects propagate to the microscale. Although similar macroscale mechanical effects of dynamic loading are present in healing tendon compared to uninjured tendon, the microscale properties differed greatly during early healing. Regression analysis identified several variables (collagen and nuclear disorganization, cellularity, and F-actin) that directly predict nuclear deformation under loading. Finite element modeling predicted deficits in ECM stress transmission following fatigue loading and during healing. Together, this work identifies the multiscale response of tendon to dynamic loading and healing, and provides new insight into microenvironmental features that tenocytes may experience following injury and after cell delivery therapies.

Vascular growth responses to chronic arterial occlusion are unaffected by myeloid specific focal adhesion kinase (FAK) deletion.

Arteriogenesis, or the lumenal expansion of pre-existing arterioles in the presence of an upstream occlusion, is a fundamental vascular growth response. Though alterations in shear stress stimulate arteriogenesis, the migration of monocytes into the perivascular space surrounding collateral arteries and their differentiation into macrophages is critical for this vascular growth response to occur. Focal adhesion kinase's (FAK) role in regulating cell migration has recently been expanded to primary macrophages. We therefore investigated the effect of the myeloid-specific conditional deletion of FAK on vascular remodeling in the mouse femoral arterial ligation (FAL) model. Using laser Doppler perfusion imaging, whole mount imaging of vascular casted gracilis muscles, and immunostaining for CD31 in gastrocnemius muscles cross-sections, we found that there were no statistical differences in perfusion recovery, arteriogenesis, or angiogenesis 28 days after FAL. We therefore sought to determine FAK expression in different myeloid cell populations. We found that FAK is expressed at equally low levels in Ly6C(hi) and Ly6C(lo) blood monocytes, however expression is increased over 2-fold in bone marrow derived macrophages. Ultimately, these results suggest that FAK is not required for monocyte migration to the perivascular space and that vascular remodeling following arterial occlusion occurs independently of myeloid specific FAK.

Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction.

OBJECTIVE: Collateral arteriogenesis, the growth of existing arterial vessels to a larger diameter, is a fundamental adaptive response that is often critical for the perfusion and survival of tissues downstream of chronic arterial occlusion(s). Shear stress regulates arteriogenesis; however, the arteriogenic significance of reversed flow direction, occurring in numerous collateral artery segments after femoral artery ligation, is unknown. Our objective was to determine if reversed flow direction in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis. APPROACH AND RESULTS: Collateral segments experiencing reversed flow direction after femoral artery ligation in C57BL/6 mice exhibit increased pericollateral macrophage recruitment, amplified arteriogenesis (30% diameter and 2.8-fold conductance increases), and remarkably permanent (12 weeks post femoral artery ligation) remodeling. Genome-wide transcriptional analyses on human umbilical vein endothelial cells exposed to reversed flow conditions mimicking those occurring in vivo yielded 10-fold more significantly regulated transcripts, as well as enhanced activation of upstream regulators (nuclear factor κB [NFκB], vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-ß) and arteriogenic canonical pathways (protein kinase A, phosphodiesterase, and mitogen-activated protein kinase). Augmented expression of key proarteriogenic molecules (Kruppel-like factor 2 [KLF2], intercellular adhesion molecule 1, and endothelial nitric oxide synthase) was also verified by quantitative real-time polymerase chain reaction, leading us to test whether intercellular adhesion molecule 1 or endothelial nitric oxide synthase regulate amplified arteriogenesis in flow-reversed collateral segments in vivo. Interestingly, enhanced pericollateral macrophage recruitment and amplified arteriogenesis was attenuated in flow-reversed collateral segments after femoral artery ligation in intercellular adhesion molecule 1(-/-) mice; however, endothelial nitric oxide synthase(-/-) mice showed no such differences. CONCLUSIONS: Reversed flow leads to a broad amplification of proarteriogenic endothelial signaling and a sustained intercellular adhesion molecule 1-dependent augmentation of arteriogenesis. Further investigation of the endothelial mechanotransduction pathways activated by reversed flow may lead to more effective and durable therapeutic options for arterial occlusive diseases.